FIG 2.

Procedure for Fe isotopic labeling of B. subtilis cells and biofilms. Cells were precultured at 30°C in liquid MSgg medium containing pure ⁵⁶Fe (10⁻⁴ M) for several generations to produce ⁵⁶Fe-labeled cells (a), and then cells were inoculated in an MSgg medium containing pure ⁵⁶Fe and grown for 22 h to produce the ⁵⁶Fe-labeled robust biofilm (b). Following biofilm formation, the ⁵⁶Fe-labeled cells and biofilm were transferred in a new MSgg medium containing no Fe (c) or pure ⁵⁷Fe, provided as FeCl₃ or Fe-tannic acid (d). Fe content (⁵⁶Fe and ⁵⁷Fe) in cells and cells plus biofilm was monitored for 6 h. Fe content in the biofilm matrix was calculated by subtracting cellular Fe from total biofilm Fe (cells plus matrix). Acquisition of new Fe was calculated by subtracting the amount of cell-bound Fe (number of cells × Fe cellular quotas) 6 h after transfer to the amount of cell-bound Fe at T₀ (composed of 100% ⁵⁶Fe).